PROJECT SUMMARY/ABSTRACT Children born to mothers with obesity are at increased risk for developing obesity and diabetes later in life, independent of lifestyle factors, such as physical activity or nutrition. While some children born to obese mothers will not go on to develop obesity or insulin resistance, for those who do, little is known about how maternal exposures (e.g., hyperinsulinemia) may influence child outcomes in humans. We use human, infant mesenchymal stem cells (MSCs), cultured from umbilical cord tissue collected at birth, for investigating mechanisms of obesity and diabetes risk in humans. Human and animal studies, as well as our own MSC data, show strong association between maternal, MSC and infant metabolism. For example, MSCs from infants born to obese vs. normal weight mothers have perturbations in lipid metabolism and energy sensing molecules regulating lipid metabolism, such as AMP-activated protein kinase (AMPK), which we observed at the epigenetic level (DNA methylation). Ob-MSCs also have impaired AMPK activation in response to metabolic stress in vitro, which could compromise lipid partitioning and shifts in fuel utilization in response to metabolic stimuli. Moreover, MSC lipid metabolism correlates with fat mass of the infants measured at birth. These observations make this a novel model in which to investigate the epigenetic programming of human metabolic phenotypes in vitro. Our unique, translational approach allows us to maintain human variability in a basic science model. Such integration of mechanistic investigation with clinical samples will help us to achieve our long-term goal of understanding mechanisms for the developmental programming of metabolism at the molecular level in humans. This project will not only advance the field for understanding the molecular underpinnings of human developmental programming, but may also identify modifiable maternal factors contributing to offspring phenotype supporting implementation of critically needed obesity prevention strategies. Therefore, the central hypothesis for this proposal is that perturbations in maternal metabolism induce infant MSC epigenetic signatures promoting dysregulation of cellular fuel switching and mitochondrial dysfunction. This project leverages our unique resource of MSCs already collected from >150 infants as part of a well characterized, longitudinal pre-birth cohort (Healthy Start, R01DK076648). Thus, while Aims 1&2 will determine mechanisms for altered lipid partitioning and mitochondrial dysfunction in MSCs from infants born to normal weight versus obese mothers, Aim 3 will expand this knowledge to population science, validating our preliminary data in a larger cohort. Aim 1 will determine the impact of maternal obesity-induced epigenetic signatures on offspring MSC fuel switching and lipid partitioning in response to changes in nutrient supply. Aim 2 will determine whether epigenetic signature promotes mitochondrial dysfunction and reduced substrate oxidation in Ob- vs. NW-MSCs. Aim 3 will substantiate the clinical relevance of the umbilical cord MSC model using specific tests to determine maternal metabolic measures predictive of MSC metabolism and, in turn, MSC metabolic measures predictive of infant outcomes.